Our goal is to estimate the frequency, location, and functional significance of nucleotide variation underlying "footprints" of adaptation in the Drosophila melanogaster genome. The proposed research builds on our successful initial scan using variation at dispersed microsatellite loci that revealed six predicted targets of selection in one region of the X-chromosome. Localized resequencing of three of these regions has uncovered positive selection affecting synonymous sites (at Notch), amino acid sequence diversification (at CG18508) and two selective sweeps putatively associated with transcription factor binding sites. Our initial analyses also revealed the need for more complete, high-quality sequence coverage to have statistical power to distinguish selection from non-equilibrium demography and to precisely localize targets of selection. We will use newly developed statistical tests for footprints of selection to analyze recently publically released (www.dpgp.org, Langley pers. Comm.) complete re-sequencing of 39 inbred lines of D. melanogaster from North American. We will also re-sequence an African sample of 50 lines for a 2 Mb region of the X-chromosome in order to estimate the timing, strength, and targets of selection in these two geographically and ecologically distinct populations. We will assay sequence variation around putative targets in twenty-two additional population samples from around the world to define the geographic regions in which the sweeps occurred. These data will provide insight into the ecological/geographic context of the selective event. Finally, we will functionally characterize three identified targets of selection. We will test hypotheses of translational regulation associated with unusual synonymous site evolution at Notch using both tissue culture and germline transformation transcription assays. We will test for a role in host- defense and/or local innate immune function at CG18508, and evaluate the functional consequences of selectively fixed Caudal transcription factor binding site difference between populations upstream of CG18508. Our results lay the foundation for understanding the frequency and molecular basis of adaptation in Drosophila, providing a benchmark against which to test our assumptions about adaptive evolution in other genomes.